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. 2021 Aug 6;187(3):1445–1461. doi: 10.1093/plphys/kiab374

Figure 5.

Figure 5

Phosphomimetic THRUMIN1 at serine 170 disrupted the filamentous localization but conferred WT chloroplast movements. A, Agrobacterium strains containing internal deletion constructs of the IDR were transiently expressed in N. benthamiana to test the localization pattern of THRUMIN1:YFP in response to blue-light stimulation. Deletion of amino acids 186–201 restored the filamentous localization of THRUMIN1. B, Serine 170, a putative phosphorylation site between amino acids 169 and 186, was mutated to alanine or aspartic acid to mimic a nonphosphorylatable or constitutively phosphorylated residue, respectively. Transient expression of 35S:THRUMIN1S170D:YFP in N. benthamiana disrupted the filamentous localization of THRUMIN1 while 35S:THRUMIN1S170A:YFP did not. Representative time-lapse images are shown for dark treatment (514 nm for YFP excitation), blue-light stimulation (470 nm and 514 nm), and then dark again. Images for transient expression of 35S:THRUMIN1S170D:YFP and 35S:THRUMIN1S170A:YFP in N. benthamiana are representative frames from Supplemental Movie S7 and S10, respectively. A and B, The scale bars indicate a 5-µm distance. Similar observations were made in at least three independent time-lapse movie replicates for each experiment shown in (A and B). C, Leaf transmittance assays testing the response to low and high blue light of Col-0 WT, thrumin1-2 mutant, 35S:THRUMIN1S170A:YFP rescue transgenic, and the 35S:THRUMIN1S170D:YFP rescue transgenic. Both mutant transgenic genotypes exhibited WT chloroplast movements. After the establishment of the baseline level of leaf light transmittance after dark acclimation, chloroplast movement was induced by treatment with low blue light (∼2 µmol m−2 s−1, low BL) followed by high blue-light intensity (∼100 µmol m−2 s−1, high BL). Standard deviation error bars represent the variance in transmittance values for 8–12 individual plants per genotype.